1. Field of the Invention
This invention relates to the regeneration of plants from excised cells, and, more particularly, to the regeneration of tobacco plants from recovered and cultured guard cells.
2. Description of the Related Art
It is possible to initiate the growth of an individual plant without the use of seeds. Such asexual reproduction may be accomplished a number of different ways. Once such way is to isolate certain types of cells from a plant, culture those cells to establish a stable population, and then promote shoot and root growth from the population to form an immature plant that can be planted in soil and grown just like a regular, seed-derived plant.
Not all types of cells in a plant can be motivated to form a new plant. Those which can are often designated as totipotent or capable of developing into a complete embryo or plant. Those which cannot are considered to be terminally differentiated and lack the ability to develop into a plant, thereby regenerating its parent. Specialized plant cells performing particular and unique functions are generally considered to be terminally differentiated. In developing their special nature, such cells may lose their totipotency.
Plant regeneration is known in the art and was achieved by Shahin (U.S. Pat. No. 4,634,674 issued Jan. 6, 1987) and Carlson, et al. (U.S. Pat. No. 3,832,801 issued Sep. 3, 1974).
In the Shahin patent, plants are regenerated from protoplasts isolated from the shoot or stem portion of pre-conditioned tomato seedlings. Shoots from germinated tomato seeds are separated from the rest of the young plant and grown in a substantially hormone free growth medium to form protoplast-donating plants. Such stems of immature plants lack any significant number of functional guard cells and do not yield guard cell protoplasts.
The Carlson, et al. patent fused mesophyll protoplasts from two Nicotiana species: Nicotiana glauca and Nicotiana sylvestris to form polyploidal hybrid cells. In performing this parasexual hybridization, Carlson, et al. used the mesophyll cells of plant leaves and discarded the epidermal layers with their guard cells.
Previously, it has been very difficult to obtain viable guard cell protoplast populations as such cells were not amenable to previously-known methods for rearing protoplasts. Often, such guard cell protoplasts would live for a limited time perhaps dividing once or twice before ceasing reproduction.
The guard cells that surround stomatal pores are specialized to transduce environmental signals into the turgor-driven cell movements that regulate plant gas exchange with the environment. Guard cells possess unique anatomical, physiological, and developmental features that reflect their function and underlie their movements. They are relatively small, have a few starch-containing chloroplasts, contain relatively high numbers of mitochondria, and have thickened, asymmetric cell walls. Unlike mesophyll cells, guard cells accumulate starch in their chloroplasts in darkness and catabolize starch in light. Guard cells extrude protons, accumulate potassium ions, and develop turgor in response to low fluences of blue light. They lose turgor when treated with abscisic acid or when subjected to high concentrations of carbon dioxide. The chloroplasts of guard cells fail to senesce (turn yellow) at the same time as chloroplasts in cells of senescing mesophyll tissue of the same leaf.
Good methods are available for preparing guard cell protoplasts free of contaminating mesophyll and epidermal cells. Such highly purified preparations containing a single cell type might be expected to exhibit homogeneous responses to cell culture protocols. Potentially, cultured guard cell protoplasts could be used to study; 1) plant cell responses to light quality and quantity; 2) cellular responses to growth regulators such as abscisic acid; 3) patterns of plant cell development, including differentiation and/or redifferentiation in culture; and 4) chloroplast senescence.
For these reasons, initiation was made of the systematic development of a method for culturing guard cell protoplasts of Nicotiana glauca (Graham), tree tobacco. This species was chosen because its leaf epidermis is easily detached, because it has large leaves with high densities of stomata, and because the genus Nicotiana is amenable to culture.
The highly specialized nature of guard cells led to examination initially as to whether they were terminally differentiated. To do so, attempts were made to establish guard cell protoplasts isolated from leaves of N. glauca in culture and to induce their division with plant growth regulators. The results of that study indicated that guard cells were not terminally differentiated and that they could be made to grow, synthesize cell walls, and divide in media similar to those used for culturing mesophyll cell protoplasts of other Nicotiana species.
The experiments leading to the present invention were undertaken for two reasons. First, the results of previous experiments suggested that optimal conditions for culturing guard cells had not been achieved. The percentage of cells surviving 96 h in culture, the time at which the first cell divisions began, was less than ten percent (&lt;10%) of the original number of cells used to initiate the culture. By day 7 of culture, survival had declined to less than one percent (&lt;1%) of the original number of cells. Although 80% of the cells surviving 7 days in culture divided, concerns arose about using callus derived from such a small number of original guard cell protoplasts to regenerate plants, since it could be argued that the surviving cells were a small subpopulation of protoplasts that were not representative of the cells of the initial isolate.
Although no morphological evidence to that effect was found, the possibility that the dividing cells might be a subpopulation that was more responsive to the peculiar culture conditions employed or that they were those guard cells most recently differentiated from progenitor cells could not be ruled out. Thus, determination was sought as to whether adjustments to culture conditions could increase the percentage of guard cell protoplasts surviving in culture.
Second, a demonstration of totipotency is usually viewed as an important step in the development of a culture protocol for a cell type which has never before been cultured. Such a demonstration is necessary to ensure that the culture conditions employed do not alter the genome to such an extent that plants cannot be recovered from callus derived from the cultured cells. Because no demonstration of totipotency has been made for cultured guard cells of any species, the hypothesis was tested that cultured guard cell protoplasts of Nicotiana glauca are totipotent by attempting to regenerate plants from callus derived from cultured guard cell protoplasts.